Project Summary/Abstract Carbohydrates cover the surface of every living cell and are the means by which cells communicate with each other. They have, therefore, enormous impact both in the healthy body as well as in many disease processes, including cancer, diabetes, heart and lung disease, AIDS, and many more. In order to understand the language of carbohydrate-mediated cell-cell communication, it is vital to know the details of glycoprotein structure. However, due to the huge diversity of carbohydrate structures this is a daunting task, and presently only a handful of laboratories in the U.S. are capable of doing this research. Detailed glycoprotein structural analysis has to be able to identify the peptide sequence where the glycans are attached, as well as the structure of the glycan portion, including oligosaccharide isomers, sequence and glycosyl linkages. Currently, mass spectrometry (MS) experiments on both released glycans as well as on intact glycopeptides are needed to fully elucidate the structure of glycoproteins. Released glycan analysis depends on a derivatization procedure called ?permethylation?, in which every hydroxyl proton is replaced by a methyl group. Separate experiments on the intact glycopeptides are needed because glycan release abolishes all site-specific information. Additional workflows are also required to separate N- from O-linked glycans and to determine degree of glycosylation. The first aim of the present proposal is to consolidate all the above workflows into one by carrying out the permethylation and MS on intact glycopeptides instead of on released glycans. This will allow all the necessary information pertaining to the glycoprotein, including glycan linkage, isomers, sequence, attachment site, and degree of glycosylation to be obtained in a one-pot experiment. The only way to expand the field of glycobiology is to make it more accessible to other scientists, and combining glycan structure elucidation with glycopeptide analysis in a single experiment is a step towards that goal. Fetuin will be used as the eukaryotic model glycoprotein and C. jejuni glycoprotein AcrA as the model for prokaryotic glycoproteins. The technology involving permethylation of glycopeptides and MSn analysis will be incorporated into the CCRC's hands-on training courses that are annually offered to the glyco-community. Hands-on training is one of the most efficient ways to make any technology assessable to the larger scientific community. In order to assist the interpretation of the complex data generated in the MS, the second aim of this project is expansion of the software generated previously in-house to include automatic annotation of permethylated glycopeptide mass spectra. The existing software was built in a modular fashion to enable the addition of further functionality through ?plugins?. The third aim is the development of a permethylation kit to facilitate the single sample preparation workflow to aid researchers who are not specialists in carbohydrate analysis.